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Development of a platform for HIV/HCV genotyping to anti-viral drug resistance using Next Generation Sequencing - NGS technology Rodrigo de Moraes Brindeiro.

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Apresentação em tema: "Development of a platform for HIV/HCV genotyping to anti-viral drug resistance using Next Generation Sequencing - NGS technology Rodrigo de Moraes Brindeiro."— Transcrição da apresentação:

1 Development of a platform for HIV/HCV genotyping to anti-viral drug resistance using Next Generation Sequencing - NGS technology Rodrigo de Moraes Brindeiro – UFRJ Amilcar Tanuri – UFRJ Mônica B. Arruda – UFRJ

2 Fazer Genotipagem do HIV usando sequenciamento de nova geração (Next Generation Sequencing – NGS) vai me deixar na moda? Mesmo achando que é caro e complexo?

3 Como usar NGS para fazer uma genotipagem completa do HIV – 5 alvos; Multiplex e de baixo custo! (e ainda agregar o HCV...)

4 Rationale(1) New ARV drugs against HIV, in new therapeutical classes, demand new genetic targets for HIV genotyping: –Integrase: Raltegravir, Elvitegravir, Dolutegravir... –env gp41: Fuzeon® T20 –env C2V3: Maraviroc,... Comercial (FDA approved) Genotyping assays/kits using Sangers modified sequencing method do not evaluate all these genetic regions (targets) where DRMs accumulate: –Just Protease (PR) and Reverse Transcriptase (RT) genes Drug resistance genotyping based on ion torrent deep sequencing can evaluate all genetic regions (targets) where DRMs accumulate, by low cost: –Protease (PR) e Reverse Transcriptase (RT) (RNAseH region included), Integrase, env gp41, env C2V3 and Gag (co-evolution of PR cutting sites and late domain)

5 Rationale(1) New ARV drugs against HIV, in new therapeutical classes, demand new genetic targets for HIV genotyping: –Integrase: Raltegravir, Elvitegravir, Dolutegravir... –env gp41: Fuzeon® T20 –env C2V3: Maraviroc,... Comercial (FDA approved) Genotyping assays/kits using Sangers modified sequencing method do not evaluate all these genetic regions (targets) where DRMs accumulate: –Just Protease (PR) and Reverse Transcriptase (RT) genes Drug resistance genotyping based on ion torrent deep sequencing can evaluate all genetic regions (targets) where DRMs accumulate, by low cost: –Protease (PR) e Reverse Transcriptase (RT) (RNAseH region included), Integrase, env gp41, env C2V3 and Gag (co-evolution of PR cutting sites and late domain)

6 Rationale(1) New ARV drugs against HIV, in new therapeutical classes, demand new genetic targets for HIV genotyping: –Integrase: Raltegravir, Elvitegravir, Dolutegravir... –env gp41: Fuzeon® T20 –env C2V3: Maraviroc,... Comercial (FDA approved) Genotyping assays/kits using Sangers modified sequencing method do not evaluate all these genetic regions (targets) where DRMs accumulate: –Just Protease (PR) and Reverse Transcriptase (RT) genes Drug resistance genotyping based on NGS sequencing can evaluate all genetic regions (targets) where DRMs accumulate, by low cost: –Protease (PR) e Reverse Transcriptase (RT) (RNAseH region included), Integrase, env gp41, env C2V3

7 Rationale (2) DRM Genotyping: not clonal, synergy between mutations not evaluated: –Syntheny between mutations multi-resistant virus or –Mutations in different subpopulations mixture of resistant and wild type viruses. ARV-resistant subpopulations present under 10-20% of total are not considered but can further impact on the therapy efficacy. The concept of depth of coverage (nber. of times a given sequence is obtained) de sequências clonais, through ion torrent sequencing, allows the evaluation of mutation occurence in viral sub-populations and thus: –Their impact on the efficacy of ARV therapy used, as well the new rescue regimen to be implemented... –Minor sub-populations carrying DRMs, circulating at the threshold level of the wild-type (ARV sensitive) major population; –The cell tropism of viral sub-populations (M-tropic x T-tropic, or CCR5 x CXCR4 tropic) when analyzing the env C2V3 region; for the correct implementation of Maraviroc therapeutics 2-5% sub-population detection sensitivity

8 Rationale (2) DRM Genotyping: not clonal, synergy between mutations not evaluated: –Syntheny between mutations multi-resistant virus or –Mutations in different subpopulations mixture of resistant and wild type viruses. ARV-resistant subpopulations present under 20% of total are not considered but can further impact on the therapy efficacy. The concept of depth of coverage (nber. of times a given sequence is obtained) de sequências clonais, through ion torrent sequencing, allows the evaluation of mutation occurence in viral sub-populations and thus: –Their impact on the efficacy of ARV therapy used, as well the new rescue regimen to be implemented... –Minor sub-populations carrying DRMs, circulating at the threshold level of the wild-type (ARV sensitive) major population; –The cell tropism of viral sub-populations (M-tropic x T-tropic, or CCR5 x CXCR4 tropic) when analyzing the env C2V3 region; for the correct implementation of Maraviroc therapeutics 2-5% sub-population detection sensitivity

9 SANGER Sequencing technologies: Not clonal: blend of populations. Do not discriminate subpopulations Not sensitive to detect mutations in minor subpopulations (under 20%)

10 Rationale (2) DRM Genotyping: not clonal, synergy between mutations not evaluated: –Syntheny between mutations multi-resistant virus or –Mutations in different subpopulations mixture of resistant and wild type viruses. ARV-resistant subpopulations present under 20% of total are not considered but can further impact on the therapy efficacy. The concept of depth of coverage (nber. of times a given sequence is obtained) of clonal sequences, through NGS sequencing, allows the evaluation of mutation occurence in viral sub-populations and thus: –Their impact on the efficacy of ARV therapy used, as well the new rescue regimen to be implemented... –Minor sub-populations carrying DRMs, circulating at the threshold level of the wild-type (ARV sensitive) major population; –The cell tropism of viral sub-populations (M-tropic x T-tropic, or CCR5 x CXCR4 tropic) when analyzing the env C2V3 region; for the correct implementation of Maraviroc therapeutics 5-10% sub-population detection sensitivity

11 Rationale(3) OTHERS: –Compatible cost.. aggregate HCV (hepatitis C) Protease and Replicase; to evaluate: viral genotype impact on therapy DRMs for replicase and protease inhibitors (Ribavirine, Boceprevir, Telaprevir,...) Um único Kit, vários alvos, dois (ou mais?) vírus...

12 Brief Today: –Genotyping of HIV by comercial methods (Sanger): Two (only) therapeutic targets Cost to MoH: aprox. U$ 125,00 to U$ 150,00 / genotyping –Genotyping HCV: No assay (in house methods, in some cases)

13 Brief Perspective NGS -Deep Sequencing: –Genotyping HIV using NGS: ALL therapeutic targets (actual: 5-6) Cost to MoH: –aprox. 1 chip (10-100Mb) multiplexed (12-20 patients/barcodes; coverage of 250x-1000x) –U$ 1.000,00 to U$ 1.500,00 / rxn = U$ 70,00 to U$ 100,00 / patient.

14 Brief Perspective Deep Sequencing: –Genotyping HCV using NGS: ALL therapeutic targets (actual: 2) Cost to MoH: –aprox. same chip (100Mb) multiplexed (12 patients/barcodes; coverage of x); OR – other chip? HBV included? TB? - Aprox cases until 2010 –Aprox in treatment –Aprox. 40% (12.000) under treatment failure

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18 R&D deep sequencing test for resistance genotyping in HIV: –R&D: Solexa illumina MiSeq –comercial kit –Roche: 454 kit, HIV PR & RT only (??) –Basic Science (inhouse) R&D – Spain (plataform 454) – Roger Paredes, PhD (group of Bonaventura Clotet, PhD MD, chief of University Hospital Germans Trias i Pujol in Barcelona, Spain )

19 Pirossequenciamento Roche 454 – GS Junior FLX

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21 Pirossequenciamento

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25 Illumina MiSeq / HiSeq

26 Illumina

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28 Ion Torrent - ionograma Platform: Ion PGM TM < Ion Proton TM

29 Ion Torrent

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31 Comparação de tecnologias

32 Chips:

33 Chips:

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35 314: –milhões de sensores –20 Mb read/coverage –Amplicons 200bp (400bp ideal next?)

36 Inicial : –7 milhões de sensores –200 Mb de cobertura total –Amplicons de 200pb (400pb ideal possível?) –Amplicons HIV: 17 (3 GAG, 2 PR, 6 RT, 3 IN, 2 ENVgp41, 1 ENVgp120) ou, 11 (2 GAG, 1 PR, 4 RT, 2 IN, 1 ENVgp41, 1 ENVgp120) –Amplicons HCV: 10 (4 PR, 6 Rep) ou, 6 (2 PR, 4 Rep) –Total: 27 ou 17

37 316: –Total: 27 ou 17 amplicons –BIDIRECIONAL (A + B-P1 adaptors para ambos primers F e R) –Capacidade: 250 amplicons com cobertura x, logo –Capacidade de detectar até 5% de variação (cut-off aceitável) – 20 a 40 leituras de 5% por região. –27amps x 200(165)pb x 1000 cob. = 5.4Mb ou –17amps x 400(365)pb x 1000 cob = 6.8Mb –5.4Mb x 18 genos/pacientes = 97.2Mb ou –6.8Mb x 14 genos/pacientes = 95.2Mb Ion DNA Barcoding 1-16 Kit (10 sets of 16 libraries) 17-n Kit

38 316: –Total: 27 ou 17 amplicons –bottleneck: »Ancoramento dos diversos primers (54 ou 34!!!) em regiões genômicas estáveis (TODAS!)... »Alguma saída?

39 316: –Total: 27 ou 17 amplicons –bottleneck: »Ancoramento dos diversos primers (54 ou 34!!!) em regiões genômicas estáveis (TODAS!)... »Alguma saída? CONTINGÊNCIA: o PLANO B!

40 314: –METHOD: AMPLICON X LIBRARY »Preparation of 2 subgenomic amplicons of HIV (~4kb: 3kb PR- RT- INT, 1kb ENV); enzyme break and library prep (adaptor ligation) »HCV: 1 amplicon (3kb PR-Replicase); enzyme break and library prep (adaptor ligation) »AB Library Builder System –Ion Xpress Fragment Library Kit (up to 20 reactions) –Ion One Touch System –PGM System

41 Library Builder & Ion One Touch

42 Long Amplicons: SuperScript III + High Fidelity Platinum Taq one step

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44 314: –Costs: »Chip: aprox. U$ 800,00 »All rxns: U$ 300,00 to U$ 500,00 »Consumables: polymerases, RT-PCR kits, plasticware, dNTP, etc. »Genotyping/patient: ~U$ 1.200/12 genos= aprox. U$ 100,00

45 Software Initial (raw data mining): CLC ® Geneious ® Proprietary ion torrent ® Other? (interpretation algorythm) Genotyping Reports: –New Development: »Language:VbNET? Delphi? Java? »Data bank: MySQL? SQL Server?

46 Calendar of Activities Submissões de projeto

47 Project Costs Plataforma Ion Torrent Kits / Chips 316;314: –Fase I: PROVA de CONCEITO treinamento e 1os testes e erros: 06 chips/kits Prova de conceito (multiplex, barcode): 04 chips/kits Testes de subtipos/genótipos/variantes:04 chips/kits Sensibilidade (LOD):10 chips/kits Sensibilidade de detecção misturas (subpopulações):04 chips/kits –Fase II: ESTUDOS CLÍNICOS de VIABILIDADE Estudo Piloto (sítio de rede labs)04 chips/kits Estudo Multicêntrico (3 sítios de rede)16 chips/kits –Mais: material de suporte (plasticaria, kits p/PCR)... –FASE II: submissão de projeto PPP: FINEP, BNDES, FAPERJ (também na FASE I final? Prova de conceito estabelecida?) –Bolsas (2) de pesquisador

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49 Perspectives New Joint Projects: 1. VL assay for HIV/HCV using digital PCR 2. Malignant Hyperthermia (Anesthesics in surgery problem) diagnostic using SNPs on digital PCR Bottlenecks: Competition - miniaturization - point-of-care: - µfluidics + - enzymes estabilization in chip = - lab-on-chip - easy-to-use credit card handle machines and iPhone devices for mol.biology!!! Lab-on-foils, lab-on-chips.

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